Constructing Mutant Ribosomes Containing Mutant Ribosomal RNAs

Chapter

Abstract

The ribosome is the factory for protein biosynthesis, consisting of 3 different ribosomal RNA (rRNA) molecules (16S, 23S, and 5S rRNAs in prokaryotes) and more than 50 different ribosomal proteins. Because almost all organisms have multiple operons for rRNA genes (rrn operons), mutational analysis of ribosomes has inevitable technical difficulties, particularly for analyzing the functions of the 16S and 23S rRNAs, which form part of the core structure for the small (30S) and large (50S) subunits, respectively. In this chapter, we introduce six major strategies that allow researchers to perform mutational studies of the prokaryotic ribosome, particularly by focusing on the analysis of the 16S and 23S rRNA molecules. Although conventional mutational studies allow only for a small number of nucleotide changes simultaneously, recent approach developed by our group circumvents this problem in the Escherichia coli 16S rRNA gene, allowing for changes of up to 20% of the total nucleotides by interspecies exchange of the gene with that from foreign (non-E. coli) bacteria. The outcome of this novel technique has led to the discovery of an unexpected, nontranslational function (ribonuclease inhibitor) in the 16S rRNA molecule. The introduction of such a large sequence perturbation into the central core of the ribosome will open up a new era of ribosomal engineering to create highly functional ribosomes or phenotypic improvements of the host cell, which would be advantageous for biotechnological applications.

Keywords

16S rRNA Mutational analysis Functional metagenomics Orthogonal (O)-translation system 

References

  1. Amunts A, Brown A, Toots J, Scheres SH, Ramakrishnan V (2015) Ribosome. The structure of the human mitochondrial ribosome. Science 348:95–98CrossRefGoogle Scholar
  2. Asai T, Zaporojets D, Squires C, Squires CL (1999) An Escherichia coli strain with all chromosomal rRNA operons inactivated: complete exchange of rRNA genes between bacteria. Proc Natl Acad Sci U S A 96:1971–1976CrossRefGoogle Scholar
  3. Baker KA, Lamichhane R, Lamichhane T, Rueda D, Cunningham PR (2016) Protein-RNA dynamics in the central junction control 30S ribosome assembly. J Mol Biol 428:3615–3631CrossRefGoogle Scholar
  4. Ban N, Nissen P, Hansen J, Moore PB, Steitz TA (2000) The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science 289:905–920CrossRefGoogle Scholar
  5. Ben-Shem A, Garreau de Loubresse N, Melnikov S, Jenner L, Yusupova G, Yusupov M (2011) The structure of the eukaryotic ribosome at 3.0 A resolution. Science 334:1524–1529CrossRefGoogle Scholar
  6. Brosius J, Dull TJ, Sleeter DD, Noller HF (1981a) Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol 148:107–127CrossRefGoogle Scholar
  7. Brosius J, Ullrich A, Raker MA, Gray A, Dull TJ, Gutell RR, Noller HF (1981b) Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon of E. coli. Plasmid 6:112–118CrossRefGoogle Scholar
  8. Brown A, Amunts A, Bai XC, Sugimoto Y, Edwards PC, Murshudov G, Scheres SH, Ramakrishnan V (2014) Structure of the large ribosomal subunit from human mitochondria. Science 346:718–722CrossRefGoogle Scholar
  9. Cannone JJ, Subramanian S, Schnare MN, Collett JR, D'Souza LM, Du Y, Feng B, Lin N, Madabusi LV, Müller KM, Pande N, Shang Z, Yu N, Gutell RR (2002) The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs. BMC Bioinformatics 3:2CrossRefGoogle Scholar
  10. Clemons WM Jr, May JL, Wimberly BT, Mccutcheon JP, Capel MS, Ramakrishnan V (1999) Structure of a bacterial 30S ribosomal subunit at 5.5 A resolution. Nature 400:833–840CrossRefGoogle Scholar
  11. Davis JH, Williamson JR (2017) Structure and dynamics of bacterial ribosome biogenesis. Philos Trans R Soc Lond Ser B Biol Sci 372:pii: 20160181CrossRefGoogle Scholar
  12. Englander MT, Avins JL, Fleisher RC, Liu B, Effraim PR, Wang J, Schulten K, Leyh TS, Gonzalez RL Jr, Cornish VW (2015) The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center. Proc Natl Acad Sci U S A 112:6038–6043CrossRefGoogle Scholar
  13. Forterre P (2015) The universal tree of life: an update. Front Microbiol 6:717CrossRefGoogle Scholar
  14. Frank J (2017) The mechanism of translation. F1000Res 6:198CrossRefGoogle Scholar
  15. Fried SD, Schmied WH, Uttamapinant C, Chin JW (2015) Ribosome subunit stapling for orthogonal translation in E. coli. Angew Chem Int Ed 54:12791–12794CrossRefGoogle Scholar
  16. Golovina AY, Bogdanov AA, Dontsova OA, Sergiev PV (2010) Purification of 30S ribosomal subunit by streptavidin affinity chromatography. Biochimie 92:914–917CrossRefGoogle Scholar
  17. Greber BJ, Boehringer D, Leibundgut M, Bieri P, Leitner A, Schmitz N, Aebersold R, Ban N (2014) The complete structure of the large subunit of the mammalian mitochondrial ribosome. Nature 515:283–286CrossRefGoogle Scholar
  18. Greber BJ, Bieri P, Leibundgut M, Leitner A, Aebersold R, Boehringer D, Ban N (2015) Ribosome. The complete structure of the 55S mammalian mitochondrial ribosome. Science 348:303–308CrossRefGoogle Scholar
  19. Green R, Noller HF (1997) Ribosomes and translation. Annu Rev Biochem 66:679–716CrossRefGoogle Scholar
  20. Gupta N, Culver GM (2014) Multiple in vivo pathways for Escherichia coli small ribosomal subunit assembly occur on one pre-rRNA. Nat Struct Mol Biol 21:937–943CrossRefGoogle Scholar
  21. Harms J, Schluenzen F, Zarivach R, Bashan A, Gat S, Agmon I, Bartels H, Franceschi F, Yonath A (2001) High resolution structure of the large ribosomal subunit from a mesophilic eubacterium. Cell 107:679–688CrossRefGoogle Scholar
  22. Jewett MC, Fritz BR, Timmerman LE, Church GM (2013) In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation. Mol Syst Biol 9:678CrossRefGoogle Scholar
  23. Kaushal PS, Sharma MR, Booth TM, Haque EM, Tung CS, Sanbonmatsu KY, Spremulli LL, Agrawal RK (2014) Cryo-EM structure of the small subunit of the mammalian mitochondrial ribosome. Proc Natl Acad Sci U S A 111:7284–7289CrossRefGoogle Scholar
  24. Kaushal PS, Sharma MR, Agrawal RK (2015) The 55S mammalian mitochondrial ribosome and its tRNA-exit region. Biochimie 114:119–126CrossRefGoogle Scholar
  25. Kitahara K, Miyazaki K (2011) Specific inhibition of bacterial RNase T2 by helix 41 of 16S ribosomal RNA. Nat Commun 2:549CrossRefGoogle Scholar
  26. Kitahara K, Miyazaki K (2014) Revisiting bacterial phylogeny. Mob Genet Elem 3(1):e24210CrossRefGoogle Scholar
  27. Kitahara K, Suzuki T (2009) The ordered transcription of RNA domains is not essential for ribosome biogenesis in Escherichia coli. Mol Cell 34:760–766CrossRefGoogle Scholar
  28. Kitahara K, Kajiura A, Sato NS, Suzuki T (2007) Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2. Nucleic Acids Res 35:4018–4029CrossRefGoogle Scholar
  29. Kitahara K, Yasutake Y, Miyazaki K (2012) Mutational robustness of 16S ribosomal RNA, shown by experimental horizontal gene transfer in Escherichia coli. Proc Natl Acad Sci U S A 109:19220–19225CrossRefGoogle Scholar
  30. Klinge S, Voigts-Hoffmann F, Leibundgut M, Arpagaus S, Ban N (2011) Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6. Science 334:941–948CrossRefGoogle Scholar
  31. Kloss P, Xiong L, Shinabarger DL, Mankin AS (1999) Resistance mutations in 23 S rRNA identify the site of action of the protein synthesis inhibitor linezolid in the ribosomal peptidyl transferase center. J Mol Biol 294:93–101CrossRefGoogle Scholar
  32. Klumpp S, Scott M, Pedersen S, Hwa T (2013) Molecular crowding limits translation and cell growth. Proc Natl Acad Sci U S A 110:16754–16759CrossRefGoogle Scholar
  33. Laios E, Waddington M, Saraiya AA, Baker KA, O’Connor E, Pamarathy D, Cunningham PR (2004) Combinatorial genetic technology for the development of new anti-infectives. Arch Pathol Lab Med 128:1351–1359PubMedGoogle Scholar
  34. Lee K, Holland-Staley CA, Cunningham PR (1996) Genetic analysis of the Shine-Dalgarno interaction: selection of alternative functional mRNA-rRNA combinations. RNA 2:1270–1285PubMedPubMedCentralGoogle Scholar
  35. Leviev IG, Rodriguez-Fonseca C, Phan H, Garrett RA, Heilek G, Noller HF, Mankin AS (1994) A conserved secondary structural motif in 23S rRNA defines the site of interaction of amicetin, a universal inhibitor of peptide bond formation. EMBO J 13:1682–1686PubMedPubMedCentralGoogle Scholar
  36. Milo R, Jorgensen P, Moran U, Weber G, Springer M (2010) BioNumbers—the database of key numbers in molecular and cell biology. Nucleic Acids Res 38:D750–D753CrossRefGoogle Scholar
  37. Miyazaki K, Sato M, Tsukuda M (2017) PCR primer design for 16S rRNAs for experimental horizontal gene transfer test in Escherichia coli. Front Bioeng Biotechnol 5:14CrossRefGoogle Scholar
  38. Moine H, Squires CL, Ehresmann B, Ehresmann C (2000) In vivo selection of functional ribosomes with variations in the rRNA-binding site of Escherichia coli ribosomal protein S8: evolutionary implications. Proc Natl Acad Sci U S A 97:605–610CrossRefGoogle Scholar
  39. Moore PB, Steitz TA (2002) The involvement of RNA in ribosome function. Nature 418:229–235CrossRefGoogle Scholar
  40. Morosyuk SV, Santalucia J Jr, Cunningham PR (2001) Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA. III. Functional analysis of the 690 loop. J Mol Biol 307:213–228CrossRefGoogle Scholar
  41. Neidhardt FC (1987) Escherichia coli and Salmonella typhimurium, cellular and molecular biology. ASM Press, Washington DCGoogle Scholar
  42. Nierhaus KH, Dohme F (1974) Total reconstitution of functionally active 50S ribosomal subunits from Escherichia coli. Proc Natl Acad Sci U S A 71:4713–4717CrossRefGoogle Scholar
  43. Noeske J, Wasserman MR, Terry DS, Altman RB, Blanchard SC, Cate JH (2015) High-resolution structure of the Escherichia coli ribosome. Nat Struct Mol Biol 22:336–341CrossRefGoogle Scholar
  44. Nomura M (1999) Engineering of bacterial ribosomes: replacement of all seven Escherichia coli rRNA operons by a single plasmid-encoded operon. Proc Natl Acad Sci U S A 96:1820–1822CrossRefGoogle Scholar
  45. Oakes M, Aris JP, Brockenbrough JS, Wai H, Vu L, Nomura M (1998) Mutational analysis of the structure and localization of the nucleolus in the yeast Saccharomyces cerevisiae. J Cell Biol 143:23–34CrossRefGoogle Scholar
  46. Orelle C, Carlson ED, Szal T, Florin T, Jewett MC, Mankin AS (2015) Protein synthesis by ribosomes with tethered subunits. Nature 524:119–124CrossRefGoogle Scholar
  47. Ott M, Amunts A, Brown A (2016) Organization and regulation of mitochondrial protein synthesis. Annu Rev Biochem 85:77–101CrossRefGoogle Scholar
  48. Quan S, Skovgaard O, Mclaughlin RE, Buurman ET, Squires CL (2015) Markerless Escherichia coli rrn deletion strains for genetic determination of ribosomal binding sites. G3 5:2555–2557CrossRefGoogle Scholar
  49. Rabl J, Leibundgut M, Ataide SF, Haag A, Ban N (2011) Crystal structure of the eukaryotic 40S ribosomal subunit in complex with initiation factor 1. Science 331:730–736CrossRefGoogle Scholar
  50. Rackham O, Chin JW (2005) A network of orthogonal ribosome x mRNA pairs. Nat Chem Biol 1:159–166CrossRefGoogle Scholar
  51. Rakauskaite R, Dinman JD (2008) rRNA mutants in the yeast peptidyltransferase center reveal allosteric information networks and mechanisms of drug resistance. Nucleic Acids Res 36:1497–1507CrossRefGoogle Scholar
  52. Ramakrishnan V (2014) The ribosome emerges from a black box. Cell 159:979–984CrossRefGoogle Scholar
  53. Rodnina MV (2013) The ribosome as a versatile catalyst: reactions at the peptidyl transferase center. Curr Opin Struct Biol 23:595–602CrossRefGoogle Scholar
  54. Saraiya AA, Lamichhane TN, Chow CS, Santalucia J Jr, Cunningham PR (2008) Identification and role of functionally important motifs in the 970 loop of Escherichia coli 16S ribosomal RNA. J Mol Biol 376:645–657CrossRefGoogle Scholar
  55. Schluenzen F, Tocilj A, Zarivach R, Harms J, Gluehmann M, Janell D, Bashan A, Bartels H, Agmon I, Franceschi F, Yonath A (2000) Structure of functionally activated small ribosomal subunit at 3.3 angstroms resolution. Cell 102:615–623CrossRefGoogle Scholar
  56. Schlünzen F, Zarivach R, Harms J, Bashan A, Tocilj A, Albrecht R, Yonath A, Franceschi F (2001) Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria. Nature 413:814–821CrossRefGoogle Scholar
  57. Schmeing TM, Ramakrishnan V (2009) What recent ribosome structures have revealed about the mechanism of translation. Nature 461:1234–1242CrossRefGoogle Scholar
  58. Schuwirth BS, Borovinskaya MA, Hau CW, Zhang W, Vila-Sanjurjo A, Holton JM, Cate JH (2005) Structures of the bacterial ribosome at 3.5 A resolution. Science 310:827–834CrossRefGoogle Scholar
  59. Shajani Z, Sykes MT, Williamson JR (2011) Assembly of bacterial ribosomes. Annu Rev Biochem 80:501–526CrossRefGoogle Scholar
  60. Shine J, Dalgarno L (1975) Determinant of cistron specificity in bacterial ribosomes. Nature 254:34–38CrossRefGoogle Scholar
  61. Terasaka N, Hayashi G, Katoh T, Suga H (2014) An orthogonal ribosome-tRNA pair via engineering of the peptidyl transferase center. Nat Chem Biol 10:555–557CrossRefGoogle Scholar
  62. Tocilj A, Schlünzen F, Janell D, Glühmann M, Hansen HA, Harms J, Bashan A, Bartels H, Agmon I, Franceschi F, Yonath A (1999) The small ribosomal subunit from Thermus thermophilus at 4.5 A resolution: pattern fittings and the identification of a functional site. Proc Natl Acad Sci U S A 96:14252–14257CrossRefGoogle Scholar
  63. Traub P, Nomura M (1968) Structure and function of E. coli ribosomes. V. Reconstitution of functionally active 30S ribosomal particles from RNA and proteins. Proc Natl Acad Sci U S A 59:777–784CrossRefGoogle Scholar
  64. Tsukuda M, Nakashima N, Miyazaki K (2015) Counterselection method based on conditional silencing of antitoxin genes in Escherichia coli. J Biosci Bioeng 120:591–595CrossRefGoogle Scholar
  65. Tsukuda M, Kitahara K, Miyazaki K (2017) Comparative RNA function analysis reveals high functional similarity between distantly related bacterial 16 S rRNAs. Sci Rep 7(1):9993Google Scholar
  66. Voorhees RM, Ramakrishnan V (2013) Structural basis of the translational elongation cycle. Annu Rev Biochem 82:203–236CrossRefGoogle Scholar
  67. Wang K, Neumann H, Peak-Chew SY, Chin JW (2007) Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion. Nat Biotechnol 25:770–777CrossRefGoogle Scholar
  68. Wimberly BT, Brodersen DE, Clemons WM Jr, Morgan-Warren RJ, Carter AP, Vonrhein C, Hartsch T, Ramakrishnan V (2000) Structure of the 30S ribosomal subunit. Nature 407:327–339CrossRefGoogle Scholar
  69. Yano K, Masuda K, Akanuma G, Wada T, Matsumoto T, Shiwa Y, Ishige T, Yoshikawa H, Niki H, Inaoka T, Kawamura F (2015) Growth and sporulation defects in Bacillus subtilis mutants with a single rrn operon can be suppressed by amplification of the rrn operon. Microbiology 162:35–45PubMedGoogle Scholar
  70. Yusupova G, Yusupov M (2014) High-resolution structure of the eukaryotic 80S ribosome. Annu Rev Biochem 83:467–486CrossRefGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.Department of Chemistry, Faculty of ScienceHokkaido UniversitySapporoJapan
  2. 2.Department of Life Science and Biotechnology, Bioproduction Research InstituteNational Institute of Advanced Industrial Science and Technology (AIST)TsukubaJapan
  3. 3.Department of Computational Biology and Medical Sciences, Graduate School of Frontier SciencesThe University of TokyoKashiwaJapan

Personalised recommendations